Inactivation kinetics of horseradish peroxidase (HRP) by hydrogen peroxide

In recent years, the peroxidase enzymes have generated wide interest in several industrial processes, such as wastewater treatments, food processing, pharmaceuticals, and the production of fine chemicals. However, the low stability of the peroxidases in the presence of hydrogen peroxide (H2O2) has limited its commercial use. In the present work, the effect of H2O2 on the inactivation of horseradish peroxidase (HRP) was evaluated. Three states of HRP (E0, E2, and E3) were identified. While in the absence of H2O2, the resting state E0 was observed, in the presence of low and high concentrations of H2O2, E2, and E3 were found, respectively. The results showed that HRP catalyzed the H2O2 decomposition, forming the species Ex, which was catalytically inactive. Results suggest that this loss of enzymatic activity is an intrinsic characteristic of the studied HRP. A model from a modified version of the Dunford mechanism of peroxidases was developed, which was validated against experimental data and findings reported by the literature.

Simulation results using these rate constants demonstrate that after a short transition period, reaction A5 is close to the equilibrium. Thus, it can be assumed that [ 2 * ] ≈ = = 1.5 10 −5 (A6) If the total radicals concentration (CT) is: Then, combining A4, A6, A7, and simplifying, the following can be obtained 2 = ( According to eq.(A9), and using the above-mentioned rate constants, the observed rate constant at pH = 9 is = 5.05 10 3 −1 −1 . This value was used for all further calculations.
Item 2. From the experimental UV-Vis spectra of the reaction mixture, the concentration of each enzyme species was obtained assuming that the absorbance of the reaction mixture at a given wavelength ( ) can be represented as the sum of absorbances corresponding to all the enzymatic species in the mixture: where , represents the extinction coefficient of the enzymatic species i at ( Fig. 1 of the manuscript).
Then, the concentration of each enzyme species was evaluated by fitting eq.(1) to the experimental UV-Vis spectrum. Figure A1 shows two typical examples of the fitting procedure;  Item 3. Figure 3 shows that under the tested conditions (HRP = 5 µM, hydrogen peroxide = 6.5 µM, pH 9), enzyme species E0 and E2 represented more than 99% of the total enzyme. Considering that initially HRP was at the resting state (E0), the initial absorbance at a given wavelength ( , ) is: However, when hydrogen peroxide is added to the reaction mixture all the enzyme is converted to E2: To improve the quality of the results it is desirable to maximize the difference between , and , (∆ ): Thus, for a given enzyme concentration ∆ only depends on the difference between 2, and 0, . Figure   A2 shows that this difference reaches a maximum at 422 nm. For this reason, this wavelength was selected to follow the interconversion between E0 and E2 as a function of time.  Table 1. Initial conditions are shown in Table A2 run2 t(s)  Figure A4. a) Full results corresponding to the peroxidatic activity (decolourization of Orange II) depicted in Figure 6. Lines represent the proposed model (R1 to R9, and eq. 2) using the coefficients shown in Table 1 Figure A6 shows that eq.(A13) represented adequately the simulated data. Fitting results (   A second set of simulations were performed to assess the effect of the initial hydrogen peroxide concentration (H2O2i) on the amount of consumed H2O2 per mol of oxidized external substrate (OII) (YH2O2/Se). In these simulations, the initial HRP and OII concentrations were 5 M, and 50 M, respectively. Then, the hydrogen peroxide yield (YH2O2/Se) was obtained as follows: where the subscripts i and t indicate the initial condition and the respective value at a given time t. Results depicted in Figure 6Ab demonstrate that YH2O2/Se increase as a function of the initial H2O2 concentration, which is in agreement with the results reported by Morales Urrea et al. (2018).

Item 5.
A pseudo-steady state approximation of the proposed model was used to calculate the fraction of each enzyme species. Because species Ex (R7 in the manuscript) is not active, we focused attention on active species E0, E1, E2, and E3. Accordingly, the active enzyme concentration (Ea) is Considering that species E0, E1, and E2 are close to a pseudo-steady state, the following expressions can be obtained: Then, the combination of eqs. (A19) and (A20) yields the following: Finally, using (A16): [ 2 ] [ 0 ] + 3 Combining (A21) with (A22) and simplifying: Equations (A26) to (A29) along with the coefficients shown in Table 1 were used to calculate the fraction of each species as a function of S, Si, and P ( Fig. 9 of the manuscript).
According to the proposed model, the consumption rate of the oxidant (RP) is given by reactions R1, R4, and R5: Then, combining eqs.(A26) to (A28) in (A30) = ( 0 0 + 1 1 + 2 2 )[ ] (A31) Figure A7 demonstrates that the presence of an external substrate (Se) enhances the consumption rate of the oxidant (RP). In particular, at low hydrogen peroxide concentrations RP in the absence of Se (black line in Fig. A7) is about 2 order of magnitude lower than RP in the presence of the substrate (red line in Fig. A7). This difference can be mainly attributted to the difference in the values corresponding to f0 in the presence and the absence of Se (see Fig. 9 in the manuscript).  Table 1 were used in all calculations.